Gangliosides are glycosylated sphingolipids with essential but enigmatic function in brain development and neural stem cell (NSC) maintenance. A critical barrier to our knowledge on ganglioside function in the brain is the absence of a systematic approach targeting key regulatory mechanisms in NSC differentiation instructed by gangliosides. Our group has pioneered research on the importance of gangliosides for growth factor receptor signaling and epigenetic regulation of NSCs. The overall goal of this project is to further elucidate the functional roles of gangliosides in NSCs based on contemporary concepts and technologies. The primary localization of glycosphingolipids (GSLs) on cell-surface microdomains and the drastic dose and composition changes during neural differentiation strongly suggest that GSLs are not only important as biomarkers but also are involved in modulating NSC functions. Many stage-specific GSL antigens in NSCs are now known, but less is understood about the mechanisms for their biological functions in modulating NSC cell fate determination. Here we will perform cellular and molecular biological analyses to elucidate the expression patterns of gangliosides, the functional roles of gangliosides in growth factor activation, and the mechanisms in regulating glycosyltransferases (GTs) during neural differentiation. The overall goal will be achieved by the following three specific aims: 1) To determine the expression of gangliosides in NSCs and the functional roles of specific gangliosides in relation to specific growth factors and their receptors for regulating NSC cell fate determination, such as self-renewal, proliferation, differentiation, migration, and survival. This will be achieved by investigation of stage-specific gangliosides in growth factor-mediated cellular events in normal and GT knockout mice; 2) To determine the regulatory mechanisms of GT expression in NSCs that account for the dramatic changes of ganglioside expression (?pathway switch?) during differentiation. In particular, we will study the post-translational regulation of GT expression by a novel enzyme complex formation mechanism at key metabolic branching points of their biosynthesis; and 3) To determine a novel epigenetic regulatory mechanism of GT expression in neuronal differentiation, particularly during postnatal neurogenesis. We will test the hypothesis that nuclear GM1 is associated with gene regulation in neuronal cells. Since GSL expression profiles are associated not only with normal development but also with pathogenic mechanisms of diseases, the proposed studies will significantly enhance the understanding of the functional role of GSLs in neurogenesis and the molecular mechanisms underlying the differential expression of stage-specific GSLs. This information will be extremely useful in providing novel strategies for disease treatment and neural regeneration by NSCs.